Refold - Insulin-like growth factor II

Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Insulin-like growth factor II
Abbreviated Name	IGF2
SCOP Family	Insulin-like
Structure Notes
Organism	Lates calcarifer (Barramundi)
UniProt Accession	O42429
SCOP Unique ID	n/a
Structure Solved
Class	Small Proteins
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	215
Molecular Weight	24645.7
Pi	9.96
Molecular Weight	24645.7
Disulphides	Unknown
Full Sequence	METQQRHGQHSLCHTCRRAESSRMKVKKMSSSSRALLFALALTLYVVEMASAETLCGGELVDALQFVCEDRGFYFSRPTSRGSNRRPQNRGIVEECCFRSCDLNLLEQYCAKPAKSERDVSATSLQVIPVMPALKQEVPRKQHVTVKYSKYEVWQRKAAQRLRRGVPAILRAKKFRRQAEKIKAQEQVIFHRPLISLPSKLPPVLLTTDNYVNHK
Notes	n/a

Expression
Report	Degger B, Richardson N, Collet C, Upton Z. (2001) General and Comparative Endocrinology, 123, 38-50
Project Aim	Expression and charactrization
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21
Expression Temp	37.0
Expression Time	4 h
Expression Vector	pGH(1-46)-VN-bIGF-II
Expression Protocol	E. coli BL21 cells transformed with [Met 1 ]-pGH(1-46)-VN-bIGF-II were grown in two 5-L fermenters (Applikon, Schiedam, NL) at 37°C, pH 7, until the absorbance of the culture at 600 nm reached 6.4 and 9.3 in the two fermenters. Cells were then induced with 0.32 M IPTG and grown for a further 4 h. Inclusion bodies containing the bIGF-II fusion protein [Met 1 ]-pGH(1-46)-VN-bIGF-II were collected from the fermentation broth by homogenisation and centrifugation as described in King et al. (1992).
Method of Induction	IPTG
Cell Density at Induction	OD 6.4 = 600
Cell Disruption Method	homogenizer
Lytic Agent	None
Pre-Refolding Purification	not specified
Solubility	insoluble

Refolding
Refolding Method	Dilution
Wash Buffer	n/a
Solubilization Buffer	8 M urea, 40 mM glycine, 0.1 M Tris, 0.5 mM ZnCl 2 , and 40 mM dithiothreitol (DTT) at pH 9.1
Refolding Buffer	4 M urea, 40 mM glycine, 0.1 M Tris, 10 mM EDTA, and 0.4 mM DTT at pH 8.5
Pre-Refolding Purification	not specified
Tag Cleaved	no
Refolding pH	8.5
Refolding Temperature	0.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	DTT
Redox Agent Concentration	0.4 mM
Refolding Protocol	Inclusion bodies (IBs) (12 g) containing the bIGF-II fusion protein were solubilized by suspension in 150 ml 8 M urea, 40 mM glycine, 0.1 M Tris, 0.5 mM ZnCl 2 , and 40 mM dithiothreitol (DTT) at pH 9.1. The dissolved and reduced inclusion bodies were desalted on a 100 mm (diameter) 350 mm column (XK50/100, Pharmacia) packed with 2 L GCL-1000 medium (Amicon, Beverly, MA) equilibrated with 8 M urea, 40 mM glycine, 0.1 M Tris, 0.5 mM ZnCl 2 , and 1.6 mM dithiothreitol at pH 9.1. A portion of the GCL-1000 eluant pool (300 mg) containing the bIGF-II fusion protein as detected by reverse-phase analytical high performance liquid chromatography (HPLC) and sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was refolded under oxidising conditions. The protein was diluted to a concentration of 0.125 mg/ml in a buffer containing 4 M urea, 40 mM glycine, 0.1 M Tris, 10 mM EDTA, and 0.4 mM DTT at pH 8.5 and the refolding reaction initiated with 1 mM 2-hydroxyethyl disulﬁde. The refolding reaction was monitored by analysis on a microbore C4 reverse-phase highperformance liquid chromatography column (2.1 mm (diameter) 100 mm, Brownlee Laboratories, Santa Clara, CA) employing a linear gradient from 25 to 60% (v/v) acetonitrile over 35 min in the presence of 0.1% (v/v) triﬂuoroacetic acid (TFA) at a ﬂow rate of 0.5 ml/min.
Refolding Assay	Unspecified
Refolding Chaperones	None
Refolding Additives	Glycine
Additives Concentration	40 mM
Refolding Yield	n/a
Purity	n/a
Notes	n/a

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